WO2014059403A1 - Protéines chimériques, compositions et procédés pour restaurer la fonction cholinestérase au niveau de synapses neuromusculaires - Google Patents

Protéines chimériques, compositions et procédés pour restaurer la fonction cholinestérase au niveau de synapses neuromusculaires Download PDF

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WO2014059403A1
WO2014059403A1 PCT/US2013/064793 US2013064793W WO2014059403A1 WO 2014059403 A1 WO2014059403 A1 WO 2014059403A1 US 2013064793 W US2013064793 W US 2013064793W WO 2014059403 A1 WO2014059403 A1 WO 2014059403A1
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protein
ache
chimeric protein
organophosphate
pharmaceutical composition
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Richard L. Rotundo
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University Of Miami
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • the present invention is directed generally to the fields of cell biology and medicine. More particularly, the present invention provides novel chimeric proteins, pharmaceutical compositions and methods for treating organophosphate poisoning in an individual by administering novel chimeric proteins that restore cholinesterase (e.g., acetylcholinesterase (AChE)) enzymatic activity.
  • novel chimeric proteins that restore cholinesterase (e.g., acetylcholinesterase (AChE)) enzymatic activity.
  • AChE acetylcholinesterase
  • Organophosphate pesticides and nerve agents act by irreversibly inhibiting the enzyme AChE at the neuromuscular junction (NMJ) and other cholinergic synapses in the central and peripheral nervous systems. More than 70,000 fatalities occur each year from exposure to organophosphate pesticides according to United Nations figures (United Nations, 2005), many of these from self administration, with more than one million related injuries (United Nations, 2004).
  • organophosphate nerve agents such as sarin and soman, and the ease with which these compounds can be synthesized and delivered, makes them potentially useful by nefarious groups such as the release of sarin in the Tokyo subway in 1995 that killed more than a dozen people and injured thousands.
  • organophosphate poisoning including nerve agent exposure.
  • Described herein are novel chimeric proteins, pharmaceutical compositions and methods for the treatment of victims (e.g., mammals such as humans) exposed to organophosphate compounds based on replacement of inactive enzyme molecules such as cholinesterases (e.g., AChE) present on accessible surfaces of target tissues including nerves, muscles and many glands. None of the currently available treatments, or even those under development, will restore function to cholinergic synapses whose AChE has been inactivated by rapidly aging organophosphate nerve agents.
  • a novel means of targeting catalytically active AChE molecules to the neuromuscular synapse for replacing the damaged AChE molecules with new ones to restore esteratic function was developed.
  • Fasciculin-2 is a 61 amino acid snake neurotoxin that binds with extremely high affinity to the peripheral anionic site of AChE.
  • Expressible plasmids encoding chimeric proteins including murine AChE fused with Fas2 at its carboxyl terminus were developed and tested. These chimeric AChE proteins were shown to bind to endogenous mammalian AChE, and attach to the sites of nerve-muscle contact in vivo. They can thus be used to replace inactivated enzyme at neuromuscular synapses in animals (including humans) exposed to organophosphates.
  • any esterase that hydrolyses acetylcholine such as butyrylcholinesterase (BuChE)
  • BuChE butyrylcholinesterase
  • Fas2 any other Fas-like molecule that can bind to the peripheral anionic site of AChE or other catalytically active esterase-like protein that can hydrolyze acetylcholine into choline and acetic acid (e.g., BuChE) with high affinity can be used.
  • protein and “polypeptide” are used synonymously to mean any peptide-linked chain of amino acids, regardless of length or post-translational modification, e.g., glycosylation or phosphorylation.
  • RNA Ribonucleic acid molecule
  • gene a nucleic acid molecule that codes for a particular protein, or in certain cases, a functional or structural RNA molecule.
  • a complementary DNA cDNA is a single-stranded DNA that is complementary to messenger RNA or DNA that has been synthesized from messenger RNA by reverse transcriptase, and can be used for expression of a particular protein.
  • nucleotide sequence means a chain of two or more nucleotides such as RNA (ribonucleic acid) and DNA (deoxyribonucleic acid).
  • patient means a mammalian (e.g., human, rodent, non-human primates, canine, bovine, ovine, equine, feline, etc.) subject to be treated and/or to obtain a biological sample from.
  • mammalian e.g., human, rodent, non-human primates, canine, bovine, ovine, equine, feline, etc.
  • bind means that one molecule recognizes and adheres to a particular second molecule in a sample or organism, but does not substantially recognize or adhere to other structurally unrelated molecules in the sample.
  • a first molecule that "specifically binds" a second molecule has a binding affinity greater than about 10 8 to 1012 moles/liter for that second molecule and involves precise "hand-in- a-glove” docking interactions that can be covalent and noncovalent (hydrogen bonding, hydrophobic, ionic, and van der Waals).
  • organophosphates refers to compounds capable of inactivating cholinesterases by phosphorylating the serine hydroxyl group located on the active site of the enzyme. Phosphorylation inactivates the enzyme when a covalent bond forms between the organophosphate molecule and the enzyme molecule.
  • exemplary organophosphates include insecticides such as malathion, parathion, diazinon, fenthion, dichlorvos, and chlorpyrifos and nerve gases such as soman, sarin, tabun, and VX.
  • DFP diisopropylfluorophosphate
  • novel chimeric proteins described herein are capable of replacing endogenous AChE.
  • "Replacing” refers to functionally replacing inactivated or otherwise damaged AChE molecules with new AChE molecules by attaching an active chimeric AChE molecule to the inactivated endogenous one to restore function.
  • labeling with regard to a probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody.
  • detectable substance i.e., physically linking
  • sample is used herein in its broadest sense.
  • a sample including polynucleotides, peptides, antibodies and the like may include a bodily fluid, a soluble fraction of a cell preparation or media in which cells were grown, genomic DNA, RNA or cDNA, a cell, a tissue, skin, hair and the like.
  • samples include saliva, serum, urine, blood and plasma.
  • treatment is defined as the application or administration of a therapeutic agent to a patient or a test animal, or application or administration of the therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease, or the predisposition toward disease.
  • safe and effective amount refers to the quantity of a component which is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention.
  • therapeutically effective amount is meant an amount of a composition of the present invention effective to yield the desired therapeutic response, for example, an amount effective to prevent or ameliorate organophosphate poisoning in an individual.
  • the specific safe and effective amount or therapeutically effective amount will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of mammal or animal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed.
  • terapéutica As used herein, the terms "therapeutic,” and “therapeutic agent” are used interchangeably, and are meant to encompass any molecule, chemical entity, composition, drug, therapeutic agent, or biological agent capable of preventing, ameliorating, or treating a disease or other medical condition.
  • chimeric proteins, compositions, kits, and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable chimeric proteins, compositions, kits, and methods are described below. All publications, patent applications, and patents mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. The particular embodiments discussed below are illustrative only and not intended to be limiting.
  • FIG. 1 is a schematic of the design of secretable green fluorescent protein (GFP) and AChE Fas2 chimeric proteins:
  • the chimeric proteins were constructed by inserting a leader sequence (LS) in front of the GFP (a) in pEGFP-Cl followed by a 15 amino acid linker peptide (L) and the humanized Fas2 coding sequence (hereinafter referred to as "GFP-Fas2").
  • LS leader sequence
  • L 15 amino acid linker peptide
  • Fas2 humanized Fas2 coding sequence
  • FIG. 2 is a series of micrographs showing binding of AChE-Fas2 to the NMJ in vivo 30 min after 5 ⁇ g intravenous (iv) injection.
  • FIG. 3A is a graph showing that injection of 5 ⁇ g of AChE-Fas2 protein rescues mice following exposure to 2x LD 50 DFP.
  • FIG. 3B is a set of photomicrographs showing that AChE activity is restored to the NMJs of mice surviving 2x LD 50 DFP after AChE-Fas2 injections.
  • compositions e.g., chimeric proteins
  • organophosphate poisoning e.g., nerve agent poisoning, pesticide poisoning
  • a subject e.g., a human subject
  • Two functional Fas2 chimeric proteins that bind to native wild type AChE were produced.
  • the marker fusion protein, GFP-Fas2 when injected into mice, homed in to the neuromuscular synapse where it bound to the endogenous enzyme and co-localized with the nicotinic acetylcholine receptor (AChR).
  • AChE nicotinic acetylcholine receptor
  • AChE-Fas2 chimeric protein that binds to the mammalian neuromuscular synapse and functionally replaces the endogenous enzyme on frozen sections of skeletal muscle and in vivo.
  • Chimeric AChE-Fas2 protein rescued mice exposed to a lethal dose of organophosphate cholinesterase inhibitor when they were injected with 2 x LD 50 DFP (2.9 ⁇ g/g BW) in saline followed 5 minutes later by an injection of 0.2 ⁇ g/g BW chimeric AChE protein or saline alone.
  • organophosphate nerve agents consisting of, in one embodiment, a targetable chimeric AChE molecule coupled to an AChE-specific snake neurotoxin, Fas2, that targets the protein to the neuromuscular synapse, thereby replacing the inactivated AChE molecules with new functional ones.
  • compositions e.g., chimeric proteins
  • methods for treating organophosphate poisoning in an individual e.g., a human subject.
  • organophosphates include nerve gases such as sarin, soman, VX and tabun, and pesticides such as malathion, parathion, diazinon, fenthion, dichlorvos, chlorpyrifos, etc.
  • the therapeutic compositions and methods of the invention (which include prophylactic treatment) in general include administration of a therapeutically effective amount of the compositions described herein to a subject in need thereof, including a mammal, particularly a human.
  • a typical chimeric protein for treating organophosphate poisoning as described herein includes a catalytically active AChE protein and a neuromuscular synapse targeting domain.
  • any catalytically active esterase-like protein that can hydrolyze acetylcholine into choline and acetic acid, e.g., butyrylcholinesterase can be used.
  • neuromuscular synapse targeting domain e.g., Fas2 domain
  • neural synapse targeting domain any peptide or protein that is capable of binding to AChE or other catalytically active esterase-like protein that can hydrolyze acetylcholine into choline and acetic acid (e.g., BuChE) at the NMJ.
  • the 61 amino acid snake toxin Fas2 was used (i.e., a Fas2 encoded by a nucleotide sequence optimized for mammalian expression) as a neuromuscular synapse targeting domain.
  • Fasciculin-1 or any other snake toxin, or derivative thereof, that binds to any catalytically active esterase-like protein that can hydrolyze acetylcholine into choline and acetic acid such as AChE or BuChE
  • the catalytically active AChE protein in a chimeric AChE-Fas2 protein as described herein lacks an oligomerization domain to make it monomeric and includes at least one mutation (e.g., a deletion, insertion, amino acid substitution) which prevents dimerization and binding of the catalytically active AChE protein to the Fas2.
  • Examples of other neuromuscular synapse targeting domains include portions of the collagenic tail, a related toxin called Fasciculin 1 (Fas- 1), from the same snake that differs by only one or two amino acids, and active peptides derived from Fas-2 or Fas-1.
  • Fasciculin 1 Fas- 1
  • Any suitable chimeric protein e.g., any chimeric AChE-Fas2 protein or BuChE-Fas2 protein that is able to bind to a neuromuscular synapse and restore AChE activity to the neuromuscular synapse in vivo after inactivation of endogenous AChE at the neuromuscular synapse can be used for treating and preventing organophosphate poisoning in an individual.
  • a human AChE or human BuChE protein is used for treating a human.
  • Human AChE and BuChE amino acid sequences are known.
  • an amino acid sequence for a human AChE protein is accession number NM_000665 and an amino acid sequence for a human BuChE is accession number NM_000055.2 (e.g., SEQ ID NO:6).
  • the amino acid sequence for a human AChE variant (variant E4-E6) is as follows:
  • MRPPQCLLHTPS LAS PLLLLLLWLLGGG VG AEGRED AELLVT VRGGRLRGIRLKTPGGP VS AFLGIPFAEPPMGPRRFLPPEPKQPWS G V VD ATTFQS VC YQ Y VDTLYPGFEGTEMWN PNRELS EDCLYLN VWTP YPRPTS PTP VLVWIYGGGFYS GAS S LD V YDGRFLVQ AERT VL VS MN YR VG AFGFLALPGS RE APGN VGLLD QRLALQW VQEN V A AFGGDPTS VTLFGES A G A AS VGMHLLS PPS RGLFHR A VLQS G APNGPW ATVGMGEARRR ATQLAHLVGCPPGG TGGNDTELV ACLRTRP AQ VLVNHEWH VLPQES VFRFS F VP V VDGDFLS DTPE ALIN AGD FHGLQVLVGVVKDEGSYFLVYGAPGFSKDNESLISRA
  • Fasciculin sequence can be used (the amino acid sequence of Fas2 is described in Bourne et al., Cell vol. 83:503-512, 1995) in the chimeric polypeptides described herein.
  • a nucleotide sequence encoding Fasciculin that has been optimized for mammalian expression is preferred.
  • Fas2 amino acid sequence (encoded by a nucleotide sequence optimized for mammalian expression) is TMC YS HTTTS RAILTNCGENS C YRKS RRHPPKM VLGRGCGCPPGDDNLEVKCCTS PDKC NY (SEQ ID NO:2).
  • a typical amino acid sequence for a chimeric AChE-Fas2 protein having a human AChE and a Fas2 sequence (encoded by a nucleotide sequence optimized for mammalian expression) includes a leader sequence followed by human AChE followed by the 15 amino acid linker sequence followed by the Fas2 sequence.
  • any variants, mutants, fragments, analogs or derivatives thereof that bind to a neuromuscular synapse and restore AChE activity to the neuromuscular synapse can be used.
  • a human AChE variant variant E4-E6
  • linker sequence linker sequence
  • Fas2 sequence a leader sequence
  • MRPPQCLLHTPS LAS PLLLLLLWLLGGG VG AEGRED AELLVT VRGGRLRGIR LKTPGGP VS AFLGIPF AEPPMGPRRFLPPEPKQPWS G V VD ATTFQS VC YQ Y VDTLYPGFE GTEMWNPNRELSEDCLYLNVWTPYPRPTSPTPVLVWIYGGGFYSGASSLDVYDGRFLV QAERTVLVSMNYRVGAFGFLALPGSREAPGNVGLLDQRLALQWVQENVAAFGGDPTS VTLFGES AG A AS VGMHLLS PPS RGLFHR A VLQS G APNGPW AT VGMGEARRRATQLAH LVGCPPGGTGGNDTELV ACLRTRP AQ VLVNHEWH VLPQES VFRFS FVPV VDGDFLS DTP EALIN AGDFHGLQ VLVG V VKDEGS YFLV YG APGFS KDNES LIS R AEFLAG VRVG VPQ
  • a nucleotide sequence encoding a chimeric AChE-Fas2 protein can be determined using the protocols and cloning details described herein.
  • Nucleic acid molecules encoding chimeric proteins as described herein include variants, such as those that encode fragments, analogs and derivatives of a chimeric AChE-Fas2 protein or chimeric BuChE-containing protein.
  • the nucleotide sequence of such variants can feature a deletion, addition, or substitution of one or more nucleotides.
  • variant chimeric AChE-Fas2 proteins or chimeric BuChE-containing proteins displaying substantial changes in structure can be generated by making nucleotide substitutions that cause less than conservative changes in the encoded polypeptide.
  • nucleotide substitutions are those that cause changes in (a) the structure of the polypeptide backbone; (b) the charge or hydrophobicity of the polypeptide; or (c) the bulk of an amino acid side chain.
  • nucleic acid molecules that have minor variations in their nucleotide sequence, by, for example, standard nucleic acid mutagenesis techniques or by chemical synthesis.
  • a chimeric protein may also include a linker peptide interposed between the catalytically active AChE protein, for example, and the neuromuscular synapse targeting domain to facilitate folding of each protein independently, and/or a sequence tag (e.g., StrepTag®, His tag, HA tag, etc.) at the amino terminus for purification of the chimeric protein.
  • a linker peptide interposed between the catalytically active AChE protein, for example, and the neuromuscular synapse targeting domain to facilitate folding of each protein independently, and/or a sequence tag (e.g., StrepTag®, His tag, HA tag, etc.) at the amino terminus for purification of the chimeric protein.
  • a pharmaceutical composition for treating or preventing organophosphate poisoning in an individual is also described herein.
  • a pharmaceutical composition typically includes a chimeric protein as described herein, and a pharmaceutically acceptable carrier, the chimeric protein present in an amount effective for restoring AChE activity to many or most neuromuscular synapses in the individual after inactivation of endogenous AChE at neuromuscular synapses due to exposure to an organophosphate (e.g., organophosphate poisoning).
  • organophosphate e.g., organophosphate poisoning
  • kits for treating or preventing organophosphate poisoning in an individual includes a therapeutically effective amount of a chimeric protein, a pharmaceutically acceptable excipient or carrier, instructions for use, appropriate packaging, and optionally, a means of administering. If desired, the kit also contains an effective amount of a therapeutic agent for ameliorating central nervous system (CNS) symptoms of organophosphate exposure (e.g., pyridostigmine, atropine and/or oximes such as 2-PAM).
  • CNS central nervous system
  • the kit includes a sterile container which contains a therapeutic or prophylactic composition; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
  • a typical method includes administering to the individual a chimeric protein or pharmaceutical composition as described herein in an amount effective to restore AChE activity to neuromuscular synapses in vivo after inactivation of endogenous AChE at neuromuscular synapses due to organophosphate exposure (poisoning).
  • the individual is typically a human, and may be suffering from respiratory failure due to exposure to the organophosphate.
  • the chimeric protein is at a concentration of about 20 ⁇ g/kg body weight to about 200 ⁇ g/kg (e.g., 19 ⁇ i ⁇ Lg, 20 ⁇ g/kg, 25 ⁇ i ⁇ Lg, 50 ⁇ g/kg, 100 ⁇ gl g, 150 ⁇ g/kg, 200 ⁇ g/kg, 205 ⁇ g/kg) in the pharmaceutical composition.
  • the pharmaceutical composition may be administered via any suitable means.
  • the chimeric protein can be administered as soon as possible after exposure to organophosphates, usually as a single intravenous dose.
  • the method can further include administering to the individual a therapeutic agent for ameliorating CNS symptoms of organophosphate exposure concomitant with or subsequent to administration of the chimeric proteins or pharmaceutical compositions described herein.
  • therapeutic agents/therapies include pyridostigmine, atropine and/or oximes such as 2-PAM (e.g., a combination of two or more of pyridostigmine, atropine and oxime).
  • 2-PAM e.g., a combination of two or more of pyridostigmine, atropine and oxime.
  • the chimeric proteins and pharmaceutical compositions described herein may be administered to a subject in need thereof in combination with one or more additional treatments as necessary. Preventing Organophosphate Poisoning In a Subject
  • chimeric proteins described above are generally for use in treating a subject who has been exposed to and/or is suffering from organophosphate poisoning
  • these or modified versions of these chimeric proteins could be used for preventing organophosphate poisoning in an individual, e.g., administering such modified proteins to an individual at risk of being exposed to organophosphate poisoning (before the individual is exposed to organophosphate poisoning).
  • the AChE protein or BuChE protein of the chimeric protein can be mutated such that it is resistant to organophosphates and pesticides.
  • the AChE protein or BuChE protein of the chimeric protein can be mutated such that it is more easily reactivated. Any chimeric proteins as described herein used for prophylaxis would be resistant to the organophosphates (e.g., nerve agents) they are designed to protect an individual from.
  • the linker and Fasciculin (e.g., Fas2) portions of the chimeric proteins described herein can be used to target other proteins to cholinergic synapses in the peripheral nervous system (PNS).
  • PNS peripheral nervous system
  • a growth factor or a trophic factor (e.g., a neurotrophin) or another molecule such as Agrin or a portion of a receptor such as LRP4 could be delivered to the NMJ.
  • any protein that when delivered to a NMJ can enhance or promote AChE protein expression, activity, NMJ formation/signaling, etc., can be delivered to NMJs using the compositions and methods described herein.
  • the chimeric proteins provided herein can be formulated into pharmaceutical compositions by admixture with pharmaceutically acceptable nontoxic excipients and carriers.
  • the formulations of the invention are useful for parenteral administration, for example, intravenous, subcutaneous, intramuscular, intraventricular, intracranial, intracapsular, intraspinal, intracisternal, or intraperitoneal administration.
  • a chimeric protein as described herein is administered orally.
  • the preferred routes of administration are intravenous, intramuscular, and subcutaneous.
  • compositions can be formulated for administration to humans or other animals (e.g., invertebrates, vertebrates such as nonhuman primates, canines, felines, bovines, etc.) in therapeutically effective amounts (e.g., amounts which eliminate or reduce the patient's or subject's pathological condition) to provide therapy for the organophosphate poisoning described above.
  • exemplary formulations include aqueous solutions or lyophilized powders for dilution.
  • the compositions may conveniently be administered in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical arts, for example, as described in Remington: The Science and Practice of Pharmacy (Remington: The Science & Practice of Pharmacy), Pharmaceutical Press; 21 edition (October 7, 2011).
  • Formulations for parenteral administration may contain as common excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, and the like.
  • the chimeric proteins and pharmaceutical compositions described herein may be in a form suitable for sterile injection.
  • the suitable active therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle.
  • acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution.
  • the aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate).
  • the chimeric proteins and pharmaceutical compositions described herein are preferably administered to a subject or patient (e.g., rodent, human) in an effective amount, that is, an amount capable of producing a desirable result in a treated subject or patient (e.g., restoring AChE function and treating or preventing organophosphate poisoning in the subject or patient).
  • Toxicity and therapeutic efficacy of the compositions utilized in methods of the invention can be determined by standard pharmaceutical procedures. As is well known in the medical and veterinary arts, dosage for any one animal depends on many factors, including the subject's size, body surface area, age, the particular composition to be administered, time and route of administration, general health, and other drugs being administered concurrently.
  • the amount of the therapeutic agent to be administered varies depending upon the manner of administration, the age and body weight of the subject, and with the clinical symptoms. Typical dose ranges would be from about 20 ⁇ g/kg to about 200 ⁇ g/kg of body weight per day.
  • the chimeric proteins and pharmaceutical compositions described herein are useful for administration to humans suffering from organophosphate poisoning characterized by, for example, inactivated AChE as described above.
  • organophosphate poisoning characterized by, for example, inactivated AChE as described above.
  • a mammal e.g., human, bovine, ovine, canine, feline, equine, rodent, etc.
  • these novel proteins effectively neutralize organophosphate poisoning by, for example, replacing inactive AChE molecules with catalytically active AChE molecules.
  • Example 1 Construction of the test GFP-Fas2 and the AChE-Fas2 chimeric proteins
  • the murine AChE has a deletion of the last 40 amino acids that form the dimerization domain, resulting in a monomeric enzyme of about 65 kDa.
  • This AChE was also modified by mutating three critical amino acids in the Fas2 binding site to produce a monomeric AChE enzyme that does not bind to Fas2 (i.e., especially its own Fas2 domain of the chimeric protein).
  • This chimeric protein was demonstrated to bind specifically to the neuromuscular junction where it restored active AChE enzyme activity to the synapse after irreversible inactivation.
  • a marker fusion protein, GFP-Fas2 was also generated and tested as described in more detail in Example 2.
  • Example 2 Replacement of inactive acetylcholinesterase molecules at the neuromuscular synapse in vivo
  • a novel approach was developed for restoring functional neuromuscular transmission following irreversible AChE inhibition using a chimeric AChE protein molecule that can home in to sites of nerve muscle contact in vivo (the NMJ) and replace the inactivated enzyme molecules.
  • This approach is capable of rescuing mice exposed to lethal doses of organophosphates when administered shortly after exposure.
  • This novel approach has the potential to protect humans from pesticide and nerve agent exposure as well as rescue them once exposed.
  • a means of targeting catalytically active AChE, or other protein molecules, specifically to the NMJ was developed.
  • Fas2 was used.
  • a chimeric fluorescent reporter protein consisting of GFP linked via a 15 amino acid linker peptide to the 61 amino acid snake toxin Fas2 (Fig. 1).
  • a leader sequence was included to specify secretion, and a 6xHis tag was added at the amino terminus to facilitate purification.
  • the construct was transfected into HEK293 cells and a stable cell line established by selection and cloning.
  • the chimeric protein was subsequently purified from defined medium or cell extracts from these cells using a nickel column and confirmed by SDS-PAGE and Western blotting.
  • Initial tests showed that the chimeric GFP-Fas2 protein bound to the neuromuscular junctions on frozen sections of mouse skeletal muscle where it co-localized with the nicotinic acetylcholine receptor.
  • mice were injected with either 2-3 ⁇ g Alexa-488 conjugated Fas2 or the chimeric GFP- Fas2 protein and sacrificed 30 minutes later.
  • the EDL and gastrocnemius muscles were dissected from the hind limbs and labeled with Alexa-555 aBtx to visualize the nicotinic receptors and the fibers teased apart and mounted on microscope slides.
  • the extracellular mammalian AChE normally exists as higher order oligomeric forms such as membrane-bound tetramers at CNS synapses or the neuromuscular junction form consisting of three tetramers covalently linked to a three stranded collagen-like tail. These forms are too large to diffuse into the synaptic space at the neuromuscular junction.
  • antibodies at around 150 kDa are still capable of diffusing into the neuromuscular synapse as this is the basis for several autoimmune diseases such as myasthenia gravis and Lambert-Eaton Myasthenic Syndrome.
  • the addition of a 15 amino acid linker (S GGGGS GGGGS GGGG (SEQ ID NO: 5)) followed by Fas2 at the carboxyl terminus, and the 8 amino acid streptavidin binding sequence at the amino terminus following the cleavable leader sequence completed the construct.
  • the advantage of inserting the StrepTag® sequence at the amino terminus is that the tag can be used for both purifying the chimeric protein by affinity chromatography as well as localizing the molecule using fluorescent StreptTactin, a genetically engineered form of streptavidin that has a higher affinity for the 8 amino acid StrepTag® sequence (IB A Biotagnology, Gottingen, Germany).
  • the construct was confirmed by sequencing and used to establish stable HEK293 cell lines secreting the chimeric protein for purification from conditioned medium.
  • mice were injected with either saline alone or containing 2 ⁇ g AChE-Fas2. The mice were sacrificed 60 minutes later, the EDL and gastrocnemius muscles dissected, labeled with Alexa-555 aBtx to visualize the nicotinic receptors and Oyster-556 StrepTactin to visualize the chimeric AChE.
  • the AChE-Fas2 protein was able to diffuse and attach to existing AChE molecules at the neuromuscular synapse in vivo.
  • the purpose of developing the targetable AChE molecule is to restore function to neuromuscular synapses inactivated by irreversible organophosphate inhibitors.
  • organophosphate AChE inhibitor DFP as a surrogate for nerve agents and pesticides. Injection of mice with 2 x LD 50 DFP followed by an intravenous injection of saline alone 5 minutes later resulted in rapid death. However injection of 5 ⁇ g of the chimeric AChE-Fas2 protein 5 minutes after injection of DFP rescued the majority of the animals.
  • the muscles of the injected mice were removed and labeled with Alexa 488-aBtx to visualize the nicotinic receptors together with the histochemical visualization of catalytically active AChE using the Karnovsky and Roots protocol. Injection of the chimeric AChE-Fas2 protein completely restored active enzyme at the neuromuscular synapse in those mice that survived.
  • the present studies demonstrate the feasibility of using a chimeric AChE-Fas2 protein to restore function to an otherwise lethally damaged neuromuscular synapse. Since the proximal cause of death following exposure to organophosphates is respiratory failure, treatment of victims with the chimeric AChE protein would be sufficient to allow recovery from the initial phase of poisoning and follow up with the more conventional treatments to ameliorate the CNS symptoms of organophosphate exposure. Although fasciculin itself is an inhibitor of AChE, the endogenous enzyme molecules to which it is attaching would already be inactivated under its conditions of use, hence that would not be a concern.
  • the effective dose of the chimeric protein is generally less than 200 ⁇ g/kg; this dose is sufficient to rescue the majority of the animals. Moreover, the amount of this protein needed to restore function to the neuromuscular synapse is much less than the amount injected because a large fraction will be bound by the cell surface AChE molecules on erythrocytes.

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Abstract

La présente invention concerne des protéines chimériques, des compositions pharmaceutiques et des méthodes de traitement de l'exposition à des organophosphates et de l'intoxication par des organophosphates (par exemple l'intoxication par un agent neurotoxique, l'intoxication par un pesticide) chez un sujet (par exemple un sujet humain) et la prévention de l'intoxication par des organophosphates chez un sujet présentant un risque d'exposition à un organophosphate. La protéine chimérique AChE-Fas2 utilisée dans les expériences décrites par les présentes s'est avérée pouvoir se lier spécifiquement à la jonction neuromusculaire in vivo, où elle restaure l'activité enzymatique à la synapse après une inactivation irréversible.
PCT/US2013/064793 2012-10-12 2013-10-14 Protéines chimériques, compositions et procédés pour restaurer la fonction cholinestérase au niveau de synapses neuromusculaires WO2014059403A1 (fr)

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CN105044270A (zh) * 2015-07-21 2015-11-11 孙国平 呼吸衰竭外周血单个核细胞蛋白质图谱模型的构建方法

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WO1995023158A1 (fr) * 1994-02-28 1995-08-31 Yissum Research Development Company Of The Hebrew University Of Jerusalem Systeme d'analyse d'un animal transgenique utilise dans la determination de substances anti-cholinesterase
WO2006076024A2 (fr) * 2004-05-14 2006-07-20 University Of Miami Procede de ciblage de molecule d'acetylocholinesterase vers la synapse neuromusculaire
US20110223188A1 (en) * 2008-08-25 2011-09-15 Solomon Langermann Targeted costimulatory polypeptides and methods of use to treat cancer

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
WO1995023158A1 (fr) * 1994-02-28 1995-08-31 Yissum Research Development Company Of The Hebrew University Of Jerusalem Systeme d'analyse d'un animal transgenique utilise dans la determination de substances anti-cholinesterase
WO2006076024A2 (fr) * 2004-05-14 2006-07-20 University Of Miami Procede de ciblage de molecule d'acetylocholinesterase vers la synapse neuromusculaire
US20110223188A1 (en) * 2008-08-25 2011-09-15 Solomon Langermann Targeted costimulatory polypeptides and methods of use to treat cancer

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VILJOEN ET AL.: "Snake Venom Toxins: THE PURIFICATION AND AMINO ACID SEQUENCE OF TOXIN Fvll FROM DENDROASPIS ANGUSTICEPS VENOM.", THE JOURNAL OF BIOLOGICAL CHEMISTRY., vol. 218, no. 14., 25 July 1973 (1973-07-25), pages 4915 - 4919 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105044270A (zh) * 2015-07-21 2015-11-11 孙国平 呼吸衰竭外周血单个核细胞蛋白质图谱模型的构建方法

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